Cyanobacterial Oscillator Hetmann Hsieh Jeffrey Lau David Ramos
Cyanobacterial Oscillator Hetmann Hsieh Jeffrey Lau David Ramos Zhipeng Sun Harvard i. GEM 2006 Background Project Goal Cyanobacteria, also known as blue-green algae, are a phylum of ancient photosynthetic bacteria. They are the simplest organisms known to possess a circadian rhythm. This rhythm is driven by the interaction of three proteins, called Kai. A, Kai. B, and Kai. C. Our goal this summer was to reconstitute the Kai oscillator in E. coli. To reconstitute the cyanobacterial Kai oscillator in E. coli We have achieved the first three of the following subgoals: 1. Create Kai. A, Kai. B, and Kai. C Bio. Bricks. 2. Combine the above with registry parts to form functional Bio. Bricks. 3. Express Kai proteins in E. coli and verify their interaction. 4. Verify oscillation in E. coli. Why care about biological oscillators in the first place? Bio-oscillators have a number of potential applications: • Regulating periodic tasks like drug delivery and pharmaceutical processes • Synchronizing biological circuits. • Aiding research into naturally-occurring oscillators A flask of delicious green cyanobacteria sits on our lab bench. A Side-by-Side Comparison: Repressilator and Cyanobacteria The repressilator, designed by Elowitz and Leibler, is a famous synthetic biological oscillator. It is driven by a triangle of mutual transcriptional repression (see below) The cyanobacteria oscillator is driven by three proteins, named Kai. A, Kai. B, and Kai. C. They are hypothesized to interact in the following way (see diagram at near right): • Kai. C exhibits spontaneous autokinase and autophosphorylase activity. • Kai. A promotes Kai. C phosphorylation and inhibits Kai. C dephosphorylation. • Kai. B inhibits Kai. A effect on Kai. C. The three Kai proteins are necessary and sufficient to create a stable oscillation in the phosphorylation state of Kai. C. The repressilator was a major achievement, but its oscillation was not very stable. As shown in the graph below, the trough of oscillation shifts upward over time while the period increases. La c Nakajima et al. , 2005 P The cyanobacteria oscillator benefits from millions of years of evolution. It has a number of desirable features: • Stability over time • Temperature compensation • Period adjustable via point mutations in Kai. C (Ishiura et al. , 1998) • Transcription-translation independence (= minimal work to reconstitute the system in E. coli) The graph on the far right demonstrates stable oscillation of the Kai clock in vitro (Nakajima et al. , 2005). These in vitro results give us confidence that the oscillator will work well in E. coli. λc. I T et Elowitz et al. , 2000 P P P Tomita et al. , 2005 Construct Creation Protein Interaction Further challenges We’ve created the following Bio. Bricks and added them to the registry: Western blot shows expression and interaction of Kai proteins. Our next step is to verify that the Kai clock is actually oscillating over time in E. coli This goal is completed by two problems of synchronization: 1 Kai genes 2 Kai genes + Lac promoter Kai. A + Kai. C Kai. B + Kai. C References M. B. Elowitz, S. Leibler, A synthetic oscillatory network of transcriptional regulators. Nature 2000 Jan 20; 403(6767) 335 -8. M. Ishiura et al. , Expression of a gene cluster kai. ABC as a circadian feedback process in cyanobacteria. Science 1998 Sep 4; 281(5382) 1519 -23 M. Nakajima et al. , Reconstitution of circadian oscillation of cyanobacterial Kai. C phosphorylation in vitro. Science 2005 Apr 15; 308(5720) 414 -5. J. Tomita et al. , No transcription-translation feedback in circadian rhythm of Kai. C phosphorylation. Science 2005 Jan 14; 307(5707) 251 -4. Lane 1: Lac + Kai. C Lane 2: Lac + Kai. B + Lac + Kai. C Lane 3: Lac + Kai. A + Lac + Kai. C 3 Procedure We transformed three of our constructs into E. coli, induced expression of the Kai genes, and sampled the cultures at 0. 55 OD. We lysed the samples and performed a Western blot with anti-Kai. C antibodies. Synchronization between cells: We need a way to synchronize the phases between cells in a culture. If the cells in a culture are out of phase, we’d expect to see no net oscillation on our Western blot (see picture below). Time Results We believe that the top band in each lane is phosphorylated Kai. C while the bottom band is non-phosphorylated Kai. C. As our model predicts, the level of phosphorylated Kai. C is higher when Kai. A and Kai. C are combined (lane 3) than when Kai. C alone is expressed (lane 1). Kai. B + Kai. C (lane 2) appears the same as Kai. C alone (lane 1), which is consistent with our model (since Kai. B has no direct effect on Kai. A). Our results verify that Kai. A and Kai. C are being expressed and interacting in E. coli. We cannot verify Kai. B protein interaction until we build our Kai. A+Kai. B+Kai. C construct, but the results from lane 2 are at least consistent with our model. Synchronization within cells: We also need to synchronize the phases of Kai. C proteins within a cell. If Kai. C is continuously produced, the newly generated proteins will be out of phase with existing proteins in the cell. We would then expect the net oscillation to decay over time. See the graph above for output from a computer model simulating this effect. Solution: We plan to solve both of these problems by pulsing the expression of the Kai genes. This will ensure that all the cells in a culture initiate expression at the same time (between-cell synchronization), and ensure that expression ceases after a short period of time (within-cell synchronization). We hope to have these experiments running soon.
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